The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells (original) (raw)

Change history

In the version of this article initially published, the description of Figure 1d,e in the first subsection of Results was incorrect; "...(Lck-Cre): _Taz_fl/fl_Lck_-Cre mice) immunized with KLH exhibited a larger TH17 population and fewer Treg cells than that of their _Taz_fl/fl littermates..." should read: "...(_Lck_-Cre)): _Taz_fl/fl_Lck_-Cre mice immunized with KLH exhibited a smaller TH17 population and more Treg cells than that of their _Taz_fl/fl littermates...". Also, the second sentence of the panel legend for Figure 1a incorrectly identified the numbers in the plots on the top row as "percent TH7 cells..."; this should read "percent TH17 cells...". These errors have been corrected in the PDF and HTML versions of this article.

In the version of this article initially published, the institution name for affiliation 3 (Maryland Anderson Cancer Center) was incorrect. The correct institution is MD Anderson Cancer Center. The error has been corrected in the HTML and PDF versions of the article.

Nat. Immunol. 18, 800–812 (2017); published online 15 May 2017; corrected after print 20 July 2017 In the version of this article initially published, the description of Figure 1d,e in the first subsection of Results was incorrect; “...(Lck-Cre): Tazfl/flLck-Cre mice) immunized with KLH exhibited a larger TH17 population and fewer Treg cells than that of their Tazfl/fl littermates.

References

  1. Korn, T., Bettelli, E., Oukka, M. & Kuchroo, V.K. IL-17 and Th17 cells. Annu. Rev. Immunol. 27, 485–517 (2009).
    Article CAS Google Scholar
  2. Littman, D.R. & Rudensky, A.Y. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140, 845–858 (2010).
    Article CAS Google Scholar
  3. Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006).
    Article CAS Google Scholar
  4. Zhou, L. et al. TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORgammat function. Nature 453, 236–240 (2008).
    Article CAS Google Scholar
  5. Xu, L., Kitani, A., Fuss, I. & Strober, W. Cutting edge: regulatory T cells induce CD4+CD25−Foxp3− T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-β. J. Immunol. 178, 6725–6729 (2007).
    Article CAS Google Scholar
  6. Yang, X.O. et al. Molecular antagonism and plasticity of regulatory and inflammatory T cell programs. Immunity 29, 44–56 (2008).
    Article CAS Google Scholar
  7. Zhou, X. et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 10, 1000–1007 (2009).
    Article CAS Google Scholar
  8. Zhou, L., Chong, M.M. & Littman, D.R. Plasticity of CD4+ T cell lineage differentiation. Immunity 30, 646–655 (2009).
    Article CAS Google Scholar
  9. Hong, J.H. et al. TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309, 1074–1078 (2005).
    Article CAS Google Scholar
  10. Kanai, F. et al. TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J. 19, 6778–6791 (2000).
    Article CAS Google Scholar
  11. Hong, W. & Guan, K.L. The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway. Semin. Cell Dev. Biol. 23, 785–793 (2012).
    Article CAS Google Scholar
  12. Zanconato, F., Cordenonsi, M. & Piccolo, S. YAP/TAZ at the roots of cancer. Cancer Cell 29, 783–803 (2016).
    Article CAS Google Scholar
  13. Varelas, X. et al. TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal. Nat. Cell Biol. 10, 837–848 (2008).
    Article CAS Google Scholar
  14. Varelas, X. et al. The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-β-SMAD pathway. Dev. Cell 19, 831–844 (2010).
    Article CAS Google Scholar
  15. Yu, F.X., Zhao, B. & Guan, K.L. Hippo Pathway in organ size control, tissue homeostasis, and cancer. Cell 163, 811–828 (2015).
    Article CAS Google Scholar
  16. Pan, D. The hippo signaling pathway in development and cancer. Dev. Cell 19, 491–505 (2010).
    Article CAS Google Scholar
  17. Harvey, K.F., Zhang, X. & Thomas, D.M. The Hippo pathway and human cancer. Nat. Rev. Cancer 13, 246–257 (2013).
    Article CAS Google Scholar
  18. Johnson, R. & Halder, G. The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat. Rev. Drug Discov. 13, 63–79 (2014).
    Article CAS Google Scholar
  19. Halder, G., Dupont, S. & Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat. Rev. Mol. Cell Biol. 13, 591–600 (2012).
    Article CAS Google Scholar
  20. Avruch, J. et al. Protein kinases of the Hippo pathway: regulation and substrates. Semin. Cell Dev. Biol. 23, 770–784 (2012).
    Article CAS Google Scholar
  21. Nehme, N.T . et al. MST1 mutations in autosomal recessive primary immunodeficiency characterized by defective naive T-cell survival. Blood 119, 3458–3468 (2012).
    Article CAS Google Scholar
  22. Abdollahpour, H. et al. The phenotype of human STK4 deficiency. Blood 119, 3450–3457 (2012).
    Article CAS Google Scholar
  23. Zhou, D. et al. The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naïve T cells. Proc. Natl. Acad. Sci. USA 105, 20321–20326 (2008).
    Article CAS Google Scholar
  24. Katagiri, K., Imamura, M. & Kinashi, T. Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat. Immunol. 7, 919–928 (2006).
    Article CAS Google Scholar
  25. Katagiri, K., Maeda, A., Shimonaka, M. & Kinashi, T. RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat. Immunol. 4, 741–748 (2003).
    Article CAS Google Scholar
  26. Katagiri, K. et al. Crucial functions of the Rap1 effector molecule RAPL in lymphocyte and dendritic cell trafficking. Nat. Immunol. 5, 1045–1051 (2004).
    Article CAS Google Scholar
  27. Katagiri, K. et al. Deficiency of Rap1-binding protein RAPL causes lymphoproliferative disorders through mislocalization of p27kip1. Immunity 34, 24–38 (2011).
    Article CAS Google Scholar
  28. Li, J. et al. Mammalian sterile 20-like kinase 1 (Mst1) enhances the stability of Forkhead box P3 (Foxp3) and the function of regulatory T Cells by modulating Foxp3 acetylation. J. Biol. Chem. 290, 30762–30770 (2015).
    Article CAS Google Scholar
  29. Dong, Y. et al. A cell-intrinsic role for Mst1 in regulating thymocyte egress. J. Immunol. 183, 3865–3872 (2009).
    Article CAS Google Scholar
  30. Du, X. et al. Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease. J. Immunol. 192, 1525–1535 (2014).
    Article CAS Google Scholar
  31. Katagiri, K. et al. Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes. EMBO J. 28, 1319–1331 (2009).
    Article CAS Google Scholar
  32. Ueda, Y. et al. Mst1 regulates integrin-dependent thymocyte trafficking and antigen recognition in the thymus. Nat. Commun. 3, 1098 (2012).
    Article Google Scholar
  33. Nishikimi, A. et al. Rab13 acts downstream of the kinase Mst1 to deliver the integrin LFA-1 to the cell surface for lymphocyte trafficking. Sci. Signal. 7, ra72 (2014).
    Article Google Scholar
  34. Tang, F. et al. The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility. Sci. Signal. 8, ra100 (2015).
    Article Google Scholar
  35. Moroishi, T. et al. The Hippo pathway knases LATS1/2 suppress cancer immunity. Cell 167, 1525–1539 (2016).
    Article CAS Google Scholar
  36. Liu, B. et al. Toll receptor-mediated Hippo signaling controls innate immunity in Drosophila. Cell 164, 406–419 (2016).
    Article CAS Google Scholar
  37. Geng, J. et al. Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity. Nat. Immunol. 16, 1142–1152 (2015).
    Article CAS Google Scholar
  38. Mou, F. et al. The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes. J. Exp. Med. 209, 741–759 (2012).
    Article CAS Google Scholar
  39. Jiao, S. et al. The kinase MST4 limits inflammatory responses through direct phosphorylation of the adaptor TRAF6. Nat. Immunol. 16, 246–257 (2015).
    Article CAS Google Scholar
  40. Raab, M. et al. T cell receptor “inside-out” pathway via signaling module SKAP1-RapL regulates T cell motility and interactions in lymph nodes. Immunity 32, 541–556 (2010).
    Article CAS Google Scholar
  41. Li, W. et al. STK4 regulates TLR pathways and protects against chronic inflammation-related hepatocellular carcinoma. J. Clin. Invest. 125, 4239–4254 (2015).
    Article Google Scholar
  42. Guo, X. et al. Single tumor-initiating cells evade immune clearance by recruiting type II macrophages. Genes Dev. 31, 247–259 (2017).
    Article CAS Google Scholar
  43. van Loosdregt, J. & Coffer, P.J. Post-translational modification networks regulating FOXP3 function. Trends Immunol. 35, 368–378 (2014).
    Article CAS Google Scholar
  44. Lei, Q.Y. et al. TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway. Mol. Cell. Biol. 28, 2426–2436 (2008).
    Article CAS Google Scholar
  45. Zhang, H. et al. TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition. J. Biol. Chem. 284, 13355–13362 (2009).
    Article CAS Google Scholar
  46. Azzolin, L. et al. YAP/TAZ incorporation in the β-catenin destruction complex orchestrates the Wnt response. Cell 158, 157–170 (2014).
    Article CAS Google Scholar
  47. Zhang, F., Meng, G. & Strober, W. Interactions among the transcription factors Runx1, RORγt and Foxp3 regulate the differentiation of interleukin 17-producing T cells. Nat. Immunol. 9, 1297–1306 (2008).
    Article CAS Google Scholar
  48. Lazarevic, V. et al. T-bet represses T(H)17 differentiation by preventing Runx1-mediated activation of the gene encoding RORγt. Nat. Immunol. 12, 96–104 (2011).
    Article CAS Google Scholar
  49. Lang, R. et al. SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunol. 4, 546–550 (2003).
    Article CAS Google Scholar
  50. Croker, B.A. et al. SOCS3 negatively regulates IL-6 signaling in vivo. Nat. Immunol. 4, 540–545 (2003).
    Article CAS Google Scholar
  51. Bendall, S.C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–696 (2011).
    Article CAS Google Scholar
  52. Amir, A.D. et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat. Biotechnol. 31, 545–552 (2013).
    Article CAS Google Scholar
  53. Wu, H. et al. The Ets transcription factor GABP is a component of the hippo pathway essential for growth and antioxidant defense. Cell Rep. 3, 1663–1677 (2013).
    Article CAS Google Scholar

Download references

Acknowledgements

We thank J. Avruch for comments on the manuscript. Supported by the National Basic Research Program (973) of China (2015CB910502 to L.C.), the National Natural Science Foundation of China (81422018 to L.C.; 31625010 and U1505224 to D.Z.; U1405225 and 81372617 to L.C.; J1310027 to D.Z.; 81472229 to L.H.; and 31600698 to J. Geng), the 111 Projects (B12001 and B06016), China's 1000 Young Talents Program (D.Z., and L.C.), the Fundamental Research Funds for the Central Universities of China-Xiamen University (20720160071 to D.Z. and 20720160054 to L.H.) and Major disease research projects of Xiamen (3502Z20149029 to L.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. Jing Geng, Shujuan Yu, Hao Zhao and Xiufeng Sun: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
    Jing Geng, Shujuan Yu, Hao Zhao, Xiufeng Sun, Ping Wang, Xiaolin Xiong, Lixin Hong, Changchuan Xie, Jiahui Gao, Yiran Shi, Jiaqi Peng, Nengming Xiao, Jiahuai Han, Dawang Zhou & Lanfen Chen
  2. Department of Laboratory Medicine, the First Affiliated Hospital, Medical College of Xiamen University, Xiamen, China
    Xun Li
  3. Department of Cancer Biology, MD Anderson Cancer Center, University of Texas, Houston, Texas, USA
    Randy L Johnson
  4. Institute of Immunology, Innovation Center for Cell Signaling Network, Zhejiang University School of Medicine, Hangzhou, China
    Linrong Lu
  5. Zhejiang University–University of Edinburgh Institute, Zhejiang University School of Medicine, Hangzhou, China
    Linrong Lu

Authors

  1. Jing Geng
    You can also search for this author inPubMed Google Scholar
  2. Shujuan Yu
    You can also search for this author inPubMed Google Scholar
  3. Hao Zhao
    You can also search for this author inPubMed Google Scholar
  4. Xiufeng Sun
    You can also search for this author inPubMed Google Scholar
  5. Xun Li
    You can also search for this author inPubMed Google Scholar
  6. Ping Wang
    You can also search for this author inPubMed Google Scholar
  7. Xiaolin Xiong
    You can also search for this author inPubMed Google Scholar
  8. Lixin Hong
    You can also search for this author inPubMed Google Scholar
  9. Changchuan Xie
    You can also search for this author inPubMed Google Scholar
  10. Jiahui Gao
    You can also search for this author inPubMed Google Scholar
  11. Yiran Shi
    You can also search for this author inPubMed Google Scholar
  12. Jiaqi Peng
    You can also search for this author inPubMed Google Scholar
  13. Randy L Johnson
    You can also search for this author inPubMed Google Scholar
  14. Nengming Xiao
    You can also search for this author inPubMed Google Scholar
  15. Linrong Lu
    You can also search for this author inPubMed Google Scholar
  16. Jiahuai Han
    You can also search for this author inPubMed Google Scholar
  17. Dawang Zhou
    You can also search for this author inPubMed Google Scholar
  18. Lanfen Chen
    You can also search for this author inPubMed Google Scholar

Contributions

J. Geng, S.Y., H.Z., X.S., P.W., X.X., L.H., J. Gao, Y.S. and J.P. performed experimental biological research; X.L. provided human blood samples; C.X. performed mass-spectrometry analysis; R.L.J. provided mutant mice; D.Z. and L.C. conceived of the project, with input from R.L.J., N.X., L.L. and J.H., co-wrote the paper; and all authors edited the manuscript.

Corresponding authors

Correspondence toDawang Zhou or Lanfen Chen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of mice with conditional knockout of MST1/2 or TAZ in T cells.

(a) Flow cytometric analysis (top panel) and quantification (lower panel) of thymocytes from 8-week-old Mst1 f/f Mst2 f/f mice or Mst1 f/f Mst2 f/f _Ox40_-Cre mice, stained with anti-CD4 and anti-CD8 antibodies. (b-d) Flow cytometric analysis (b) and quantification of CD3+CD4+ or CD3+CD8+ T cell (c), the naïve T cell (CD62LhighCD44low) and the effector T cell (CD62LlowCD44high) (d) populations in the spleen or lymph node from Mst1 f/f Mst2 f/f mice and Mst1 f/f Mst2 f/f _Ox40_-Cre mice, with the indicated antibodies. (e and f) Photograph of 10-month-old _Mst1_-/- KO mice with Sjögren’s syndrome (e, left panel). Representative H&E-stained eye sections with associated lymphocytic cell infiltration of the lacrimal gland (e, right panel). Relative mRNA levels of Taz, Ifng, Il17a, Il17f and Il23r genes in the lacrimal gland by Real-time quantitative PCR (f). Scale bars, 500μm. The data are representative of three independent experiments with similar results (a-f). The data represent mean ± s.d. (n=3). ns, not significant (P > 0.05); *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control, Student’s t-test.

Supplementary Figure 2 Characterization of T cells from _Taz_f/f_Lck_-Cre mice.

(a) Flow cytometric analysis (top panel) and quantification (lower panel) of thymocytes from 8-week-old Taz f/f mice or Taz f/f _Lck_-Cre mice, stained with anti-CD4 and anti-CD8 antibodies. (b-d) Flow cytometric analysis (b) and quantification of B220+ B cells, CD3+CD4+ or CD3+CD8+ T cell (c), the naïve T cell (CD62LhighCD44low) and the effector T cell (CD62LlowCD44high) (d) populations in the spleen or lymph node of Taz f/f mice and Taz f/f _Lck_-Cre mice, with the indicated antibodies. (e-f) Flow cytometry analysis of the activation markers CD69, CD25, CD62L, and CD44 on cell surfaces (e) and enzyme-linked immunosorbent assay of IL-2 or IFNγ production (f) in culture medium of CD4+ T cells isolated from 8-week-old Taz f/f mice or Taz f/f _Lck_-Cre mice, stimulated with anti-CD3 or anti-CD3 plus anti-CD28 antibodies for 24 hours. Data were assessed with Student’s t-test and are represented as mean ± s.d. ns, not significant. (g) CFSE proliferation assay of CD4+ T cells isolated from 8-week-old Taz f/f mice or Taz f/f _Lck_-Cre mice, stimulated with anti-CD3 plus anti-CD28 antibodies for 48 hours. (h) CFSE proliferation assay of Th17 or Treg cells differentiated from Taz f/f mice or Taz f/f _Lck_-Cre naïve T cells, stimulated with anti-CD3 plus anti-CD28 antibodies for indicated time. (i and j) Quantification of intracellular or ELISA assay of IL-17F expression (i) or Real-time quantitative PCR analysis of the expression levels of indicated genes in CD4+ naïve T cells from Taz f/f mice or Taz f/f _Lck_-Cre mice differentiated under TH17. (k and l) H&E (k) and anti-CD3 (l) staining for lymphocyte infiltration in the spinal cord of Taz f/f mice and Taz f/f _Lck_-Cre mice subjected to EAE by MOG immunization. Scale bars, 100μM. (m and n) Proliferation rate (m) and IL-2 production (n) of responder CD4+ naïve T cells cocultured with CD4+CD25+ Treg cells from Taz f/f mice or Taz f/f _Lck_-Cre mice and wild-type irradiated spleen cells and stimulated with anti-CD3 antibodies. The data are representative of three independent experiments with similar results. The data represent the mean ± s.d. (n=3). ns, not significant (P > 0.05); *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control, Student’s t-test.

Supplementary Figure 3 Smad3 and STAT3 synergistically upregulate Taz expression.

Identification of Smad3 and STAT3 by mass spectrometry in streptavidin-precipitation assays using biotinylated Taz gene 1-kb promoter (Taz p1000) DNA fragments in lymphoid tissue lysates of WT mouse. The control DNA was amplified from PGL3-Basic vectors using the same primer set. The DNA gel in the bottom shows the input of biotinylated DNA fragments. M, molecular size markers. The data are representative of three independent experiments with similar results.

Supplementary Figure 4 TAZ directly binds to RORγt and enhances its transcriptional activity.

(a) Identification and the list (the right panel) of TEAD3, TEAD4, RORγt, Foxp3 and Tip60 by mass spectrometry in a Flag-tagged TAZ-precipitation assay in lymphoid tissue lysates of WT or Taz Tg mice. *, migration of TAZ; IP, immunoprecipitation; IB, immunoblot analysis; M, molecular size markers. (b, c) Immunoassays of 293T cells expressing various combinations (above lanes) of GFP-tagged RORγt and full-length (FL) or the truncated fragment of Flag-TAZ as indicated; immunoprecipitation with anti-Flag and analysis by immunoblot with the indicated antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. (d) Immunoassays (as in b, c) of 293T cells expressing various combinations (above lanes) of GFP-tagged TAZ and full-length (FL) or the truncated fragment of Flag-RORγt as indicated. (e) Super-resolution immunofluorescence imaging (SIM) of HeLa cells transfected with nuclear localization sequence (NLS)/Flag-tagged TAZ (red), full-length, DBD, Hinge or LBD fragment of RORγt (green). Scale bars, 20 μm. The data are representative of three independent experiments with similar results (a-e), or with ~50 cells (e)

Supplementary Figure 5 TAZ blocks acetylation of Foxp3 and promotes its degradation.

(a) Real-time quantitative PCR (RT-qPCR) analysis of the expression levels of Foxp3 in CD4+ naïve T cells cultured with anti-CD3/CD28 antibodies in the presence of TGF-β (2.5 ng/ml) or TGF-β plus IL-6 (30 ng/ml) for 1 and 2 days. Data were assessed with Student’s _t_-test and are represented as mean ± s.d. ns, not significant. (b) Immunoassays of 293T cells expressing various combinations (above lanes) of HA-tagged Tip60, and full-length (FL) or the truncated fragment of Flag-TAZ as indicated; immunoprecipitation with anti-Flag and analysis by immunoblot with the indicated antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. (c) Immunoassays (as in b) of 293T cells expressing various combinations (above lanes) of GFP-tagged TAZ, full-length (FL) or the truncated fragment of Flag-Tip60 as indicated. (d) Immunoassays (as in b) of 293T cells expressing various combinations (above lanes) of GFP-tagged Foxp3, and full-length (FL) or the truncated fragment of Flag-Tip60 as indicated. (e) Immunoassays (as in b) of 293T cells expressing various combinations (above lanes) of Myc-tagged Tip60, Flag-Foxp3 and increasing doses of HA-tagged TAZ. (f) SIM of HeLa cells transfected with NLS/Flag-tagged TAZ (purple), HA-tagged Tip60 (red) or Flag-Foxp3 (green); Scale bars, 20 μm. The data are representative of three independent experiments with similar results (a-f), or with ~50 cells (f).

Supplementary Figure 6 TAZ activates RORγt by blocking the inhibitory effect of Foxp3 on RORγt.

(a) Immunoassays of 293T cells expressing various combinations (above lanes) of GFP-tagged Foxp3, and full-length (FL) or the truncated fragment of Flag-TAZ as indicated. immunoprecipitation with anti-Flag and analysis by immunoblot with the indicated antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. (b) Immunoblot analysis (as in a) of Foxp3 and various forms of TAZ in total lysates (bottom) and anti-Flag immunoprecipitates (top) from 293T cells expressing GFP-tagged Foxp3 and Flag-tagged TB-WW or the TB fragment of TAZ. (c) Immunoassays (as in a) of 293T cells expressing various combinations (above lanes) of GFP-tagged TAZ and full-length (FL) or the truncated fragment of Flag-Foxp3 as indicated. (d) Immunoassays (as in a) of 293T cells expressing various combinations (above lanes) of GFP-tagged RORγt, mCherry-tagged Foxp3WT or Foxp3LL/AA and Flag-tagged TAZ. (e) SIM of HeLa cells transfected with NLS/Flag-tagged TAZ (purple), mCherry-tagged Foxp3 (red) and GFP- RORγt (green); Scale bars, 20 μm. (f) A proposed working model showing dimerized/polymerized TAZ required for the assembly of the Foxp3-TAZ-RORγt complex. The data are representative of three independent experiments with similar results (a-e), or with ~50 cells (e).

Supplementary Figure 7 TEAD sequesters TAZ from the TH17 master regulator RORγt to promote the development of Treg cells.

(a) Immunoassays of 293T cells expressing various combinations (above lanes) of Flag-tagged RORγt, Foxp3 or TEAD1 and HA-tagged TAZ; immunoprecipitation with anti-Flag and analysis by immunoblot with the indicated antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. (b) Immunoassays (as in a) of 293T cells expressing various combinations (above lanes) of Flag-tagged TAZWT or TAZS51A, GFP-tagged TEAD1 or increasing doses of GFP-tagged RORγt. (c) SIM of HeLa cells cotransfected with NLS/Flag-tagged TAZWT or TAZS51A (purple), HA-tagged TEAD1 (red) or GFP-tagged Foxp3 (green); outlined areas are enlarged 4× in the corner insets. Scale bars, 20 μm. (d) Immunoassays (as in a) of 293T cells expressing various combinations (above lanes) of Flag-tagged TAZ, HA-tagged Tip60 or increasing doses of HA-tagged TEAD1. (e) Immunoassay (as in a) of 293T cells expressing various combinations (above lanes) of HA-tagged Tip60, HA-tagged p300, Flag-tagged Foxp3 and/or GFP-tagged TAZ or HA-tagged TEAD1, treated with DMSO or the histone deacetylase inhibitors Trichostatin A and Nicotinamide (TSA&NAM). immunoprecipitation with anti-Flag and analysis by immunoblot with anti-K-Ace (α-K-Ace), anti-Flag (α-Flag), anti-HA (α-HA) or anti-GFP (α-GFP); below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation.

(f) Immunoblot analysis of TEAD1 in CD4+ naive T cells infected with a retrovirus expressing shRNA for knocking down TEAD1 or control shRNA, differentiated under Treg -polarizing conditions. The data are representative of three independent experiments with similar results (a-f), or with ~50 cells (c).

Supplementary Figure 8 A proposed working model showing TAZ-mediated regulation of TH17 or Treg cell differentiation.

TAZ potentiates TH17 differentiation through direct transcriptional activation of RORγt and promoting Foxp3 degradation by reducing Tip60-mediated acetylation of Foxp3, while under Treg-skewing conditions, upregulated TEAD1 sequesters TAZ from RORγt, Tip60 and Foxp3, thereby negatively regulating TAZ-mediated TH17 differentiation but promoting Treg differentiation.

Supplementary information

Rights and permissions

About this article

Cite this article

Geng, J., Yu, S., Zhao, H. et al. The transcriptional coactivator TAZ regulates reciprocal differentiation of TH17 cells and Treg cells.Nat Immunol 18, 800–812 (2017). https://doi.org/10.1038/ni.3748

Download citation